Streptomyces plasmid and culture

ABSTRACT

Novel Streptomyces plasmids have the characteristic that their presence in non-integrated form in a micro-organism of the species Streptomyces lividans confers on that micro-organism the properties (a) of forming &#34;pocks&#34; when grown on a &#34;lawn&#34; of that strain of micro-organism deposited with the National Collection of Industrial Bacteria (NCIB) under the reference number 11416, and (b) of not forming &#34;pocks&#34; when grown on a &#34;lawn&#34; of that strain of micro-organism deposited with the NCIB under the reference number 11417, or which is derivable from a plasmid having such a characteristic by the removal or addition of DNA therefrom. 
     The plasmids are prepared from micro-organisms containing them or by the manipulation of other plasmids of the group and are of value as vectors for the introduction of nucleic acid into micro-organisms.

This invention relates to the incorporation of nucleic acid intocellular systems, to vectors for effecting such incorporation and tomicro-organisms containing such vectors.

Methods for the incorporation of foreign nucleic acid into cellularsystems have recently received much attention, the resultant modifiedcells being of interest either as a means of producing the foreignnucleic acid through replication of the cells or through the impartingof valuable properties to the cells by virtue of the presence of theforeign nucleic acid therein.

Much of the previous work in this area has involved the use ofEscherichia coli as the host cellular system for the foreign nucleicacid but we have turned our attention instead to the genus Streptomycesand related genera, and have identified a novel group of plasmids ofparticular value as vectors for use with such micro-organisms.

Accordingly the present invention comprises a Streptomyces plasmid whichhas the characteristic that its presence in non-integrated form in amicro-organism of the species Streptomyces lividans confers on thatmicro-organism the properties (a) of forming "pocks" when grown on a"lawn" of that strain of micro-organism deposited with the NationalCollection of Industrial Bacteria (NCIB) under the reference number11416, and (b) of not forming "pocks" when grown on a "lawn" of thatstrain of micro-organism deposited with the NCIB under the referencenumber 11417, or which is derivable from a plasmid having such acharacteristic by the removal or addition of DNA therefrom.

Plasmids having the characteristic defined above constitute a relatedfamily isolable as covalently closed circular (ccc) DNA which possess aportion of their DNA sequence in common and thereby possess certaincommon properties throughout the family. The properties include theformation of "pocks" when a micro-organism of the species S. lividanscontaining a plasmid of this family in non-integrated form is grown on a"lawn" of the micro-organism NCIB 11416. The term "lawn" is employedherein, as in common usage, to indicate a confluent culture. The term"pocks" indicates foci of inhibition of growth of the cells constitutingthe lawn resulting from inhibition by the cells growing thereon; thisphenomenon also being referred to as "tramlining" in relation to largerareas of inhibition. Such inhibition is believed to be analogous to thelethal zygosis described in the case of E. coli, being hereinafterreferred to in this way, and is believed to result from a transfer ofthe plasmid into the cells forming the lawn from those growing on it.

The micro-organism NCIB 11416 is a strain of S. lividans which willfunction as a passive partner against which strains containing anon-integrated plasmid of this family will express lethal zygosis. Thisis believed to be because in NCIB 11416 a plasmid of this family capableof functional expression is absent although the strain is believed tocontain such a plasmid in a form in which it is not expressible,presumably due to some type of integration. In contrast, themicro-organism NCIB 11417 is a strain of S. lividans which contains aplasmid of the family in non-integrated form and functionallyexpressible form. The presence of such a plasmid not only confersresistance against lethal zygosis initiated by a strain of S. lividanscontaining that plasmid but it has also been found that theinter-relation between plasmids of this family is such that lethalzygosis does not occur when a strain of S. lividans containing a plasmidof the family is grown on a lawn of a strain containing that plasmid orany other plasmid of the family in functionally expressible form.

It will be appreciated that the present invention extends to plasmids asdefined above in isolated form, i.e. when not present in amicro-organism, or when present in a micro-organism. The invention thusincludes a micro-organism containing such a plasmid. Also included bythe invention is a culture system comprising a micro-organism containinga plasmid as defined herein together with a culture medium therefor.Such a medium will generally be a synthetic or artificial one althoughcertain of the ingredients incorporated therein may of course be ofnatural occurrence. Various media described in the art for the cultureof Streptomyces are suitable but among these R2 medium is of particularinterest. The micro-organism will most usually be a strain ofStreptomyces lividans but may also be a Streptomyces of another speciesparticularly since the plasmids may be transferable from an S. lividansstrain to a strain of another species by crossing as describedhereinafter. Micro-organisms of particular interest are those in whichthe plasmid is present in functionally expressible form in terms of theexhibition or not of lethal zygosis in relation to two appropriatelyselected strains of the same species, and accordingly the presentinvention especially includes a micro-organism of the speciesStreptomyces lividans characterised by the properties (a) of forming"pocks" when grown on that strain of micro-organism deposited with theNCIB under reference number 11416, and (b) of not forming "pocks" whengrown on that strain of micro-organism deposited with the NCIB under thereference number 11417.

Furthermore, the invention especially includes a Streptomyces plasmidwhenever isolated from such a micro-organism as just defined. However,it has been found that the plasmid may readily be isolated from only aproportion of those micro-organisms fulfilling the functional tests forthe presence of a plasmid. Isolation is conveniently effected, and theability of a micro-organism to yield the plasmid is conveniently testedfor, by lysis of the cells of the organism, for example using an enzymesuch as lysozyme and a surface active agent such as sodium dodecylsulphate (SDS), followed by separation of the DNA constituting theplasmid from chromosomal DNA, for example by caesium chloridecentrifugation in the presence of ethidium bromide.

A group of micro-organisms exemplifying a range of plasmids according tothe present invention has been deposited with the NCIB on May 25, 1978in respect of NCIB 11417 (and also 11414, 11415 and 11416 which arereferred to herein), on Apr. 19, 1979 in respect of 11499 and 11500, andon May 24, 1979, in respect of 11501, 11502 and 11503..sup.(1) The NCIBreference numbers accorded to these micro-organisms are indicated inTable 1 given below, together with the corresponding John Innesreference numbers for these micro-organisms and the plasmids isolabletherefrom. Such micro-organisms, and other micro-organisms of thisinvention, are of special interest when in a biologically purified form,i.e. free from at least a proportion of other strains of micro-organismwith which they are in admixture in their original occurrence (naturalor otherwise). The micro-organisms are preferably in biologically pureform, i.e. essentially free from other strains of micro-organism,particularly of other Streptomyces. However, on storage, a proportion ofthe cells of strains containing a plasmid may spontaneously lose theplasmid so that they are then present in admixture with a proportion ofstrains not containing the non-integrated plasmid. The biological purityof such micro-organisms is then qualified by the presence of such"revertant" micro-organisms. It is of course possible, if desired, torepurify the original strain after such "reversion".

                  TABLE 1                                                         ______________________________________                                        Strain of S. lividans  Plasmid contained                                      Reference number               John Innes                                     NCIB       John Innes  DSM     Reference number                               ______________________________________                                        11417      M170        1568      SLP1.1.sup.(i)                               11499      M180        1569    SLP1.2                                         11500      M183        1570    SLP1.3                                         11501      M221        1571    SLP1.4                                         11502      M222        1572    SLP1.5                                         11503      M223        1573    SLP1.6                                         ______________________________________                                         .sup.(i) Designated SLP1 in U.K. Patent Application No. 26219/78.        

The micro-organism deposited under the NCIB reference number 11416 (JohnInnes Institute reference number 1326) referred to above has the sametaxonomy as the wild type strain which is described in the article byKrasilnikov et al, The Biology of Certain Groups of Actinomycetes(editor Krasilnikov), published by Science Press, Moscow, 1965, 74, atpages 109 and 110 thereof and, indeed, is believed to be the strainoriginally isolated by Krasilnikov. The micro-organisms deposited underthe NCIB reference numbers 11417, 11499, 11500, 11501, 11502 and 11503again have the same taxonomy as the wild type strain but differ from itin that each contains an autonomous plasmid separate from thechromosome. The present invention particularly includes these specificmicro-organisms and the plasmids isolable therefrom.

The derivation of these micro-organisms is as follows. Themicro-organisms NCIB 11417, 11499 and 11500 were obtained by the cultureof certain of the strains constituting the pocks arising on culture ofthe micro-organism NCIB 11416 on plates of R2 medium. Such pocks arisefrom the presence of a very low level of occurrence of variant strainsamong the micro-organisms constituting the type strain NCIB 11416. Themicro-organisms NCIB 11501, 11502 and 11503 were obtained by means of agenerally applicable procedure which comprises crossing a S. lividansmicro-organism containing a non-integrated plasmid of this family withanother species of Streptomyces such as S. coelicolor or S. parvulus andthen crossing the resulting Streptomyces of the other species withanother S. lividans micro-organism such as NCIB 11416. It is not clear,however, whether such crossing procedures give rise directly to thestrains containing the new plasmids of the family or whether thepresence of these plasmids in the S. lividans strains used in thecrosses merely becomes apparent during the manipulations used in thecrossing procedures. The specific procedures used for these threemicro-organisms were as follows. A cross of the S. lividans strain NCIB11499 with the S. coelicolor strain NCIB 11414 gave the S. coelicolorstrain of John Innes reference number M200 and this was followed by afurther cross of M200 with S. lividans NCIB 11416 which gave a group ofstrains, two of which were NCIB 11502 and 11503. A second, similarprocedure involved a cross of the S. lividans strain NCIB 11417 with theS. coelicolor strain 11414 to give the S. coelicolor strain of JohnInnes reference number M171 followed by a further cross of M171 with S.lividans NCIB 11416 to give a group of strains, one of which was NCIB11501. S. coelicolor strains M200 and M171 were selected by theirability to show lethal zygosis against a lawn of the passive S.coelicolor strain 11414, the phenomenon being a general one amongstrains of the same species of Streptomyces for other species as well aslividans. As indicated above, the crossing procedures specificallydescribed may be varied both with regard to the S. lividans strainsused, substituting another S. lividans strain such as one of strainsNCIB 11500, 11501, 11502 and 11503 for NCIB 11499 or NCIB 11417 oranother S. lividans strain not expressing the plasmid for NCIB 11416,and to the S. coelicolor strains used, substituting another S.coelicolor strain such as the strain NCIB 11415 for NCIB 11414 orsubstituting a strain of a third Streptomyces species such as S.parvulus for NCIB 11414.

The six plasmids SLP1.1 to SLP1.6, which are sex factors, each appear tobe self-replicating and self-transmissible, and have been found to be ofvarying size ranging from an estimated 6.25 megadaltons for SLP1.6 to anestimated 8.23 megadaltons for SLP1.2, and to contain varying numbers ofsites for cleavage by various restriction endonucleases. The structureof the plasmids is described in more detail in Example 1 at page 18 to19 and is summarised in Table 2 thereof. It is believed that thepresence of non-integrated plasmids of this family such as SLP1.1 toSLP1.6 in micro-organisms such as NCIB 11417 and 11499 to 11503 arisesthrough the splitting out or copying of part of the sequence ofintegrated nucleic acid in the micro-organism (although it is believedthat chromosomally integrated plasmid also is present in strains such asthese). Moreover, it is believed that the portion of DNA therebytransferred from integrated to non-integrated form is variable,containing a common portion which dictates the properties of the plasmidas defined hereinbefore together with a variable portion of thechromosomal DNA. Such a hypothesis explains the varying size of theplasmids SLP1.1 to SLP1.6 and the existence therein of a common portionof DNA as indicated in Table 2.

It will be appreciated that in addition to variations in the plasmids ofthe family described herein which are believed arise from the operationof the phenomenon described above in their in vivo generation, it isalso possible to manipulate a plasmid in vitro to produce another memberof the family by techniques such as cleavage with a restrictionendonuclease followed by ligation which are described hereinafter inmore detail in relation to the use of the plasmids as vectors. By suchtechniques one may remove portions of the DNA constituting the plasmidwhich are not vital to the functional expression of lethal zygosis asdescribed hereinbefore. On the basis of the hypothesis advanced above,such DNA clearly includes chromosomal DNA but may also include certainportions of the DNA common to all plasmids generated without any invitro involvement in their history, since not all of this may benecessary to the expression of lethal zygosis. Plasmids of the familymay also be produced by the incorporation of extra DNA through in vitromanipulation as defined hereinafter.

In summary, therefore, plasmids according to the present invention maybe obtained by various means, including directly from naturallyoccurring micro-organisms, from micro-organisms obtained by manipulationof such naturally occurring micro-organisms, and by the in vitromanipulation of plasmids obtained from either of such sources.Accordingly, it will be appreciated that the term "Streptomyces plasmid"as used herein does not imply any limitation that the plasmid isobtained directly from a Streptomyces micro-organism containing it.

The identity of such plasmids produced by in vitro manipulation, orindeed of any plasmid, as being one of the family according to thepresent invention may be ascertained through the transformation into thestrain of micro-organism deposited with the NCIB under reference number11416, using techniques described hereinafter, and the subsequent studyof the properties of the new strain thereby produced with respect topock formation on NCIB 11416 and NCIB 11417. The transformed NCIB 11416strain will, as an alternative criterion if required, no longer act aspassive partner against which a strain such as NCIB 11417 shows lethalzygosis. The procedures involved in pock detection are specificallyexemplified in Example 2. In general, cells of the strain to be testedfor active expression of lethal zygosis are cultured on a suitablemedium, for example R2 medium, together with cells of the strain whichis the passive partner, the latter being at a sufficient density toprovide a confluent lawn. After a suitable period of growth, usuallyfrom 2 to 7 days, for example 3 days, pock formation against the lawnwill occur in appropriate instances. In the event that pocks do notappear, growth is conveniently continued until the lawn sporulates, theoccurrence of sporulation without pock formation providing a sufficientindication of the absence of the lethal zygosis phenomenon.

Furthemore, it will be appreciated that although the lethal zygosisphenomenon provides a ready method of identifying a plasmid according tothe family covered by the present invention, it is nevertheless possiblethat valuable plasmids may be produced by the removal of such a portionof their DNA content that the plasmid no longer expresses the lethalzygosis phenomenon but retains the ability to replicate, or even thatthe addition of DNA could have such an effect under certaincircumstances. Accordingly the present invention includes such derivedplasmids which do nevertheless contain a portion of their DNA sequencein common with other members of the group, and which may alternativelybe defined as constituting plasmids which comprise that portion of theDNA sequence of the plasmid referred to herein as SLP1.6, this being theplasmid which is obtainable from the micro-organism deposited with theNCIB under No. 11503, which is required for replication. It will beappreciated from the foregoing discussion that such a plasmid may belarger or smaller than SLP1.6 and may be in isolated form or in amicro-organism, particularly a strain of S. lividans, especially innon-integrated form.

The presence of cleavage sites for various restriction enzymes inplasmids according to the present invention such as SLP1.1 to SLP1.6renders the plasmids of particular interest as vectors for nucleic acid,particularly DNA, inserted at such a site or sites in the plasmid. Ofespecial interest in this respect are plasmids containing a singlecleavage site for one particular enzyme, so that SLP1.1 and SLP1.5 areof interest as possessing a single site for SalPI (.tbd.PstI) whilstSLP1.2 is of interest as possessing a single site for BamHI. Theparticular value of these sites, as compared with the single site forEcoRI in all of the plasmids, is that they occur in the extra segment ofthese plasmids not present in SLP1.6 and accordingly the insertion ofDNA therein should not damage functions essential for plasmidreplication.

The insertion of foreign or additional DNA into the plasmids may beeffected, for example, by using recent developments in recombinant DNAtechnology such as those described by Collins, Current Topics inMicrobiology and Immunology, 1977, 78, 121 and also described in otherareas of the quite broad area of art now existing in relation to thesetechniques. The method used, which is exemplified hereinafter in theExamples, conveniently comprises digesting the plasmid with anappropriate restriction endonuclease to effect cleavage at a target sitetherein. Digestion may be effected at one site only in which case theDNA of the original plasmid is retained in the final plasmidincorporating the additional DNA or at more than one site in appropriatecircumstances since it may be possible to discard certain of the orginalportion of the plasmid DNA whilst still retaining appropriate propertiesin the final plasmid constituted of part of the original DNA togetherwith the foreign DNA. Following digestion, the cleaved plasmid DNA isconveniently joined to the additional DNA by ligation, for example usinga DNA ligase, the additional DNA conveniently having been tailored tohave suitable ends for ligation to the cleaved ends of the plasmid.Isolation of the reconstituted plasmid is then conveniently effected byscreening for clones which can be identified as containing thisreconstituted plasmid, for example by a fractionation procedure based onsize and/or through the identification of an inherent property in theplasmid or more conveniently of a marker introduced therein through theadditional DNA such as resistance to a particular antibiotic. As analternative, a crude or at least a partially purified mixture oforiginal and reconstituted plasmids may be used in the next stage.

As indicated above, the plasmids of the present invention provide a verysuitable means for introducing additional DNA into a micro-organism.More usually, such additional DNA will include DNA introduced into theplasmid by the in vitro manipulation thereof, although plasmids isolatedfrom natural sources may also be introduced without further manipulationif so required. Usually, however, the DNA introduced will be at least inpart foreign to the host micro-organism. Although incorporation intomicro-organisms of other genera, for example related Actinomycetes suchas Streptosporangium,Actinoplanes, and especially Nocardia andMicromonospora may be considered, the main area of interest lies withthe Streptomyces. Conveniently, such incorporation is effected by theuse of a suitable transformation method although other procedures suchas transduction and conjugation may be used. Incorporation by suchprocedures is well described in the art. Among the various methodsavailable for effecting transformation which are described in the art,one is of particular interest. This method comprises the uptake of theDNA, which is generally in covalently closed circular form, byprotoplasts obtained from the host micro-organism, various methodsdescribed in the art being suitable for the production of suchprotoplasts, for example the use of lysozyme, particularly followingtreatment with glycine. Among methods for inducing protoplasts to takeup DNA we have found that the use of polyethylene glycol (PEG) givesparticularly good results. A preferred concentration range of PEG in themedium used is from 5 or 10 to 50 or 70% w/v, particularly from 10 to30% w/v, for example 20% w/v. Following incubation of the protoplastswith the DNA the protoplasts are cultured on a suitable regenerationmedium for cell wall formation and growth, such as R2 medium,conveniently using serial dilution in application to the medium.

Following transformation, or other procedure for incorporating theplasmid, it is of course necessary to detect and isolate microorganismswhich result from its incorporation. A particularly suitable procedurefor doing this relies upon the phenomenon of lethal zygosis andcorresponds essentially to the first functional test for identifying aplasmid of the family according to this invention as described above,i.e. the formation of pocks on NCIB 11416. The cells obtained at the endof the incorporation procedure, for example on the regeneration ofprotoplasts, may represent mixtures or single colonies depending on thedilution level, etc. One of two procedures is therefore usuallyemployed. Thus, either a mixture of cells is used in the assay procedurein serial dilutions, or samples of single colonies are used. In eithercase the identification of a colony of a micro-organism incorporating aplasmid of the family is made by the occurrence of pocks. Where such acolony is applied singly in the first instance, the original colony maythen be cultured further to produce the micro-organism in quantity, butwhere the colony is derived from a mixture, then a sample may be removedfrom the centre of the " pock" on the lawn and cultured to producequantities of the micro-organism. The latter procedure is that which ismore generally used in cloning procedures.

The incorporation of plasmids according to the invention which arecapable of replication but not of exhibiting lethal zygosis is of coursenot amenable to detection in this particularly convenient manner andsuch a procedure is of course also only applicable where the originalmicro-organism does not contain a plasmid according to the inventionwhich itself produces lethal zygosis against a lawn of NCIB 11416. Itwill be appreciated that the present invention particularly includesmicro-organisms containing a plasmid as defined herein when incorporatedtherein by manipulative procedures as described above and particularlyby transformation.

Although the plasmids can be used as vectors for various forms offoreign DNA and for the purpose of producing quantities of the DNA bycultivation of the micro-organism into which it has been introduced, thearea of particular interest involves the production of antibiotics.Various species of Streptomyces can produce antibiotics in culture,including S. coelicolor, S. rimosus, S. venezuelae and S. clavuligerus,and DNA may be transferred between certain different strains or speciesusing the plasmids according to the present invention with the broad aimof strain improvement, particularly the improvement of characteristicsof the micro-organism which are relevant to industrial fermentation, forexample the production of new antibiotics, increased yields ofantibiotics, improved fermentation characteristics etc. Specifically,such procedures include the introduction of genes coding for variouspotential antibiotic side-chains or for the addition or removal offunctional groups on the molecules, as well as the introduction of genescoding for appropriate hydrolytic enzymes to allow the culture to begrown on cheaper carbohydrate sources or the amplification of geneproducts involved in rate-limiting biosynthetic steps.

The invention is illustrated by the following Examples.

EXAMPLE 1: PREPARATION OF PLASMID

The preparation from a micro-organism of a plasmid of the presentinvention is divided into the steps of identifying and isolating amicro-organism containing such a plasmid and then isolating the plasmidfrom the micro-organism. A generally applicable description is given inrelation to both steps with particular reference to strains which havebeen subjected to these general procedures and to specificmicro-organisms and plasmids isolated thereby.

ISOLATION OF MICRO-ORGANISM (A) Direct Procedure

Spores of the S. lividans strain NCIB 11416 are spread on plates of R2medium (Hopwood and Wright, Molecular and General Genetics, 1978, 162,307) and grown thereon, when some pocks are observed. Using a fineneedle, growth is picked from the centre of a pock and streaked out on aplate of the minimal medium described by Hopwood in BacteriologicalReviews, 1967, 31, 373 (MM) to give single isolated colonies. These aregrown up and then replica plated using a velvet pad on to a plate of R2medium spread with a lawn of spores of the strain NCIB 11416. Colonieson the original plate which give rise to tramlines on the replica plateare thereby identified. Such a colony is transferred with a needle on toa plate of the minimal medium (MM) and cultured to provide a quantity ofthe particular S. lividans strain containing a plasmid of the presentinvention.

Using this procedure the S. lividans strains NCIB 11417, 11499 and 11500were isolated.

(B) Procedure involving crossing

(1) A cross is made by mixing together spores of the S. lividans strainNCIB 11499, which contains the plasmid SLP1.2, and spores of the S.coelicolor strain NCIB 11414 on a slant of the complete medium describedby Hopwood, ibid (CM) and growing the mixture of spores thereon. Thespores are harvested and plated on R2 medium plates containing histidineand uracil (for growth of S. coelicolor) and streptomycin (forinhibition of S. lividans), and spread with a lawn of spores of the S.coelicolor strain NCIB 11414. Growth on these plates gives some pockswhich are picked with a needle as described in (A) and streaked out on aplate of the minimal medium (MM) containing histidine, uracil andstreptomycin to give single isolated colonies. These are grown up andthen replica plated on to a plate of R2 medium containing histidine,uracil and streptomycin, and spread with a lawn of spores of the strainNCIB 11414. Colonies on the original plate which give rise to tramlineson the replica plate are thereby identified. Such a colony istransferred with a needle on to a plate of the minimal medium containinghistidine, uracil and streptomycin and cultured to provide a quantity ofthe particular S. coelicolor strain showing the lethal zygosisphenomenon.

Using this procedure the S. coelicolor strain having the John Innesreference number M200 was isolated; it did not prove possible toseparate plasmid DNA from this strain.

(2) A cross is made by mixing together spores of the S. coelicolorstrain M200 showing the lethal zygosis phenomenon with spores of the S.lividans strain NCIB 11416 on a slant of the complete medium (CM) andgrowing the mixture of spores thereon. The spores are harvested byplating on R2 medium plates containing no histidine or uracil (forinhibition of S. coelicolor) and no streptomycin (for growth of S.lividans), and spread with a lawn of spores of the strain NCIB 11416.Growth on these plates gives rise to some pocks which are treated asdescribed under (1) above but with the omission from the media ofhistidine, uracil and streptomycin. This procedure yields particular S.lividans strains containing a plasmid of the present invention.

Using this procedure for the crossing of the S. coelicolor strain ofreference number M200 with the S. lividans strain NCIB 11416 the S.lividans strains NCIB 11502 and 11503 were isolated.

Using an analogous procedure the S. lividans strain NCIB 11417 whichcontains the plasmid SLP1.1 was crossed with the S. coelicolor strainNCIB 11414 to give the S. coelicolor strain having the John Innesreference number M171, which was in turn crossed with the S. lividansstrain NCIB 11416 leading to the isolation of the S. lividans strainNCIB 11501.

ISOLATION OF PLASMID (All percentages quoted are weight/volume.)

500 ml of a liquid medium containing 0.3% Difco Bacto yeast extract,0.5% Difco Bacto peptone, 0.3% Oxoid malt extract, 1% glucose, 34%sucrose and 0.1% magnesium chloride is inoculated with 1 ml of a densespore suspension of the micro-organism and this is grown at 30° C. withconstant gyratory shaking for 44 hours. The mycelium is harvested bycentrifugation for 30 minutes at 10,000 rpm in a Beckman JA14 rotor at20° C.

The mycelium is washed in 10% w/v glycerol, resuspended in 50 ml of0.01M Tris-HCl and 0.001M EDTA buffer of ph 8.0 (TE buffer) containing34% sucrose, and maintained at 30° C. 10 ml of 0.25M EDTA (ethylenediamine tetra-acetic acid, Na salt), pH 8.0 is added followed by 5 mllysozyme (50 mg/ml in 0.01M Tris). The mixture is incubated at 30° C.for 15 minutes, placed in an ice bath and treated with 50 ml of TEbuffer containing 34% sucrose, 30 ml of 0.25M EDTA and 5 ml of 0.01MTris (each of these being ice cold). Sodium dodecyl sulphate (SDS)dissolved in TE buffer is added to give a final concentration of 1%followed by sodium chloride to a final concentration of 1M. The lysismixture is allowed to stand in ice for 2 hours or overnight and thechromosomal DNA/SDS precipitate is removed by centrifugation at 16,000rpm and 4° C. for 1 hour in a Beckman JA20 rotor. Polyethylene glycol6000 (PEG) is then added to the supernatant to give a finalconcentration of 10% and the mixture left for 2 hours or overnight at 4°C.

The flocculent DNA-PEG precipitate is harvested by centrifugation at4,000 rpm for 4 minutes at 4° C. in a Beckman JA14 rotor and thenredissolved in the minimum volume of 0.03M Tris, 0.005M EDTA and 0.05MNaCl buffer of pH 8.0 (TES buffer) (ca 8 ml). Ethidium bromide is addedto a final concentration of 500 μg/ml and caesium chloride to arefractive index of 1.3925 (density 1.63 g/cc). The gradients arecentrifuged in a Beckman 40 rotor at 36,000 rpm and 20° C. for 60 hoursin a Beckman L2-65B centrifuge. The plasmid DNA is removed from thegradient by the careful insertion of a 1 ml syringe. Ethidium bromide isremoved from the plasmid DNA by extracting three times with two volumesof ice-cold isoamyl alcohol. Caesium chloride is removed by dialysisagainst three changes of 1000 volumes TE buffer at 4° C. for 6 hours.The yield of DNA is typically from 10 to 30 μg depending on the strain.

Using the procedure described above the six micro-organisms NCIB 11417,11499, 11500, 11501, 11502 and 11503 yielded respectively the plasmidsSLP1.1, SLP1.2, SLP1.3, SLP1.4, SLP1.5 and SLP1.6. The cleavage sites ofthe plasmid DNA were studied in the usual manner for the restrictionendonucleases EcoRI(E), HindIII (H), SalGI (G), BamHI (B), and SalPI orPstI (P). The plasmids were found to contain a common segment of DNA ofestimated molecular weight 6.25×10⁶ daltons or 6.25 megadaltonscontaining one site for cleavage by EcoRI, two sites for cleavage byHindIII and two sites for cleavage by SalGI. All of the plasmids withthe exception of SLP1.6 were found to contain an additional segmentcontaining from one to five cleavage sites. The cleavage sites in theSLP1.6 plasmid are shown in the accompanying Figure together with theestimated distances between them in megadaltons. The additional segmentin the other five plasmids is in each case inserted into the portion ofthe SLP1.6 plasmid which is represented by a thickened line. Details ofthese segments are given in the following Table 2. The estimated sizeswere obtained by comparison of the cleavage fragments obtained withstandard cleavage fragments obtained from bacteriophage lambda asmeasured at Stanford University, Calif., U.S.A. using agarose gelelectrophoresis.

                  TABLE 2                                                         ______________________________________                                        Estimated   Restriction Sites                                                                           Extra Segment                                       Plasmid                                                                             Size Md   E     H   G   B   P     .sup.(1)                              ______________________________________                                        SLP1.6                                                                              6.25      1     2   2   0   0   None                                    SLP1.4                                                                              6.4       1     3   2   0   0                                                                                  ##STR1##                               SLP1.3                                                                              6.88      1     3   3   0   0                                                                                  ##STR2##                               SLP1.1                                                                              7.13      1     3   3   0   1                                                                                  ##STR3##                               SLP1.5                                                                              7.28      1     3   3   0   1                                                                                  ##STR4##                               SLP1.2                                                                              8.23      1     3   3   1   2                                                                                  ##STR5##                               ______________________________________                                         .sup.(1) Inserted from left hand to right hand end in clockwise fashion       into portion of SLP1.6 shown as thickened line in the Figure.            

EXAMPLE 2: TRANSFORMATION OF A STREPTOMYCES WITH AN SLP PLASMID (a)Transformation Procedure

0.2 ml of a suspension of approximately 10⁷ protoplasts (prepared fromthe S. lividans strain NCIB 11416 by the method of Hopwood et al,Nature, 1977, 268, 171 but using 1 mg/ml of lysozyme alone rather than amixture of lysozyme and Lytic Enzyme No. 2) in medium P (Hopwood andWright, Molecular and General Genetics, 1978, 162, 307) is centrifugedfor 7 minutes. After pelleting the protoplasts, the supernatant isdiscarded and the protoplasts resuspended in the small volume ofremaining medium P by gently tapping the bottom of the tube with afinger. Approximately 2 μg of SLP DNA (ca 7×10¹⁰ molecules--preparedfrom an SLP⁺ strain as described in Example 1) in a small volume ofmedium P are added to the protoplasts and the tube slowly rotated toensure thorough mixing. 0.5 ml of a solution of 20%(w/v) polyethyleneglycol (PEG) 1000 in P medium is added and efficient mixing of theprotoplasts and PEG achieved by drawing the total volume up and down aPasteur pipette twice. After 1 minute, 4 ml of medium P are added andthe protoplasts are pelleted by centrifugation at 1000 g for 7 minutes.The supernatant is discarded and the protoplasts again resuspended inthe small volume of remaining liquid by gently tapping the tube with afinger. 0.2 ml of medium P is then added and serial dilutions of themixture are made by taking 20 μl and diluting successively into 0.18 mlof medium P down to a 10⁻⁵ dilution. Using a 1 ml pipette, each of thesuccessive dilutions is transferred to plates of R2 medium (Hopwood andWright, ibid), the protoplasts being spread gently over the surface ofthe plates with a wire loop or glass spreader. Regeneration of theprotoplasts and subsequent growth to produce mature sporulatingconfluent lawns or, in the case of the more dilute samples, singlecolonies, is allowed to occur for a period of 7 to 10 days at 30° C.

(b) Detection of Transformation

To assay for transformation even at low frequencies, the regenerationplates containing confluent lawns of regenerated protoplasts are takenand the spores harvested from each as follows. 10 ml of steriledistilled water is added to the plate and the surface of the culturegently scraped with a wire loop to remove the spores. The liquid is thentransferred to a small bottle and shaken vigorously to break up thespore chains into single spore units. The preparation is then filteredthrough cotton wool to remove mycelial debris and the resulting sporesuspension concentrated by centrifugation at 1000 g for 15 minutes. Thesupernatant is discarded and the remaining spore pellet resuspended in0.3 ml of 20% v/v glycerol. Serial dilutions of the preparation are madedown to 10⁻⁵ by successive transfer of 0.1 ml of the spore suspension to0.9 ml of 20% v/v glycerol. The spores can then be stored at -20° C.with little loss of viability. 0.1 ml aliquots of some of the dilutionseries (e.g. 10°, 10⁻², 10⁻⁴) of each of the harvested plates are thentransferred to R2 medium plates which have also received sufficientspores of the NCIB 11416 strain originally used in the transformationprocedure to produce a confluent lawn. Transformants can typically bedetected after 3 days' growth at 30° C. by the appearance of "pocks"resulting from "lethal zygosis" expressed by spores containing theplasmid in expressible form within the lawn.

(c) Recovery of Transformants

The transformants are recovered by picking spores from the centre of the"pock" using a fine wire needle to an agar plate of minimal medium(Hopwood, Bacteriological Reviews, 1967, 31, 373).

Varying levels of transformation have been observed in experiments withthe plasmids SLP1.1, SLP1.2, SLP1.3, SLP1.4, SLP1.5 and SLP1.6, rangingfrom at least above 1% for SLP1.3 to SLP1.6, through typical figures of18, 10, 23, 17 and 15% for SLP1.1 to as high as 85% for SLP1.2 (thepercentages referring to that proportion of the recipient protoplastscontaining at least one plasmid per protoplast).

We claim:
 1. A Streptomyces plasmid which has the characteristic that itcomprises that portion of the DNA sequence of the plasmid, designatedSLP 1.6 and shown in the Figure, required for the expression ofreplication and that its presence in non-integrated form in amicroorganism of the species Streptomyces lividans confers on thatmicroorganism the properties (a) of forming "pocks" when grown on a"lawn" of that strain of microorganism deposited with the NCIB under thereference number 11416, and (b) of not forming "pocks" when grown on a"lawn" of that strain of microorganism deposited with the NCIB under thereference number 11417, said plasmid being in isolated form.
 2. AStreptomyces plasmid selected from the group consisting of the plasmidsisolable, respectively, from the strains of microorganism deposited withthe NCIB under the reference numbers 11417, 11499, 11500, 11501, 11502,and 11503, said plasmid having the structural features given in thefollowing Table

    ______________________________________                                                                     Extra Segment                                                                 (Inserted from left hand                                                      to right hand end in                                    Esti-   Restriction Sites for                                                                       clockwise fashion into                                  mated   the Endonucleases                                                                           portion of SLP 1.6                               Plas-  Size    E, H, G, B, P shown as thickened line                          mid    Md      E     H   G   B   P   in the FIG.)                             ______________________________________                                        SLP 1.6                                                                              6.25    1     2   2   0   0   None                                     SLP 1.4                                                                              6.4     1     3   2   0   0                                                                                  ##STR6##                                SLP 1.3                                                                              6.88    1     3   3   0   0                                                                                  ##STR7##                                SLP 1.1                                                                              7.13    1     3   3   0   1                                                                                  ##STR8##                                SLP 1.5                                                                              7.28    1     3   3   0   1                                                                                  ##STR9##                                SLP 1.2                                                                              8.23    1     3   3   1   2                                                                                  ##STR10##                               ______________________________________                                    

and being in isolated form.
 3. A Streptomyces plasmid which comprisesthat portion of the DNA sequence of the plasmid, designated SLP 1.6 andshown in the Figure, required for the expression of replication andlethal zygosis, said plasmid being in isolated form.
 4. A biologicallypure culture of a microorganism containing a plasmid as defined in claim1, 2 or 3 in non-integrated form, wherein the microorganism is of thespecies Streptomyces lividans.
 5. A biologically pure culture of amicroorganism of the species Streptomyces lividans characterized by theproperties (a) of forming "pocks" when grown on a "lawn" of that strainof microorganism deposited with the NCIB under the reference member11416, and (b) of not forming "pocks" when grown on a "lawn" of thatstrain of microorganism deposited with the NCIB under the referencenumber
 11417. 6. A biologically pure culture of a strain of Streptomyceslividans selected from the group consisting of those strains depositedwith the NCIB under the reference numbers 11417, 11499, 11500, 11501,11502 and 11503.